RNA interference (RNAi) is an endogenous pathway in eukaryotic cells for reducing the expression of a target mRNA through the introduction of complementary double-stranded RNA (dsRNA). The discovery of RNAi has provided a means for analysis of biological networks and identification of therapeutic targets. For reasons of immunogenicity, RNAi in mammalian cells is initiated by short interfering RNAs (siRNAs). Despite the intense study to date on the RNAi pathway and the use of siRNAs, the identification of the most active sequences and efficient delivery of those sequences to the cells of interest remain significant challenges. It is proposed here to address these challenges of siRNA selection and delivery through analysis of multiple steps in the RNAi pathway. The proposed work will focus on two approaches to optimizing siRNA function. First, the binding interactions of siRNAs with important RNAi pathway proteins, will be characterized. Each of the proteins is known to be central to the RNAi pathway, but their contributions to silencing are not fully-understood. Second, the interactions of siRNAs with delivery vehicles built from chemically-diverse oligomeric and polymeric nanoparticles will be quantitatively analyzed to determine those structural features that encourage complex formation, protection of the siRNAs from degradation, and release of siRNAs upon entry into the cell. The polymeric nanoparticles to be studied are readily modified to provide a means of creating, with exquisite control, a diverse array of vehicles that we will use to test variables such as amine density, polyethylene glycol modification, hydrophilicity, and binding cooperativity. The overall goal of the proposed research is to design siRNAs with maximal function through manipulation of the siRNA structure and sequence and the design of vehicles with optimal chemical and physical characteristics. PUBLIC HEALTH RELEVANCE: RNA interference (RNAi), a technique for blocking the expression of a specific protein, has the potential to be an important therapeutic strategy. To realize this potential with maximum safety and activity, RNAi-based therapeutics must be designed to have optimal function for a variety of steps in their complex mechanism of action. The proposed work will address two of these important steps to provide guidelines for the design of highly-active, highly-specific therapeutics based on RNAi.